WO1991005555A2

WO1991005555A2 - N-acyl peptide metalloendopeptidase inhibitors and methods of using same

Info

Publication number: WO1991005555A2
Application number: PCT/US1990/005904
Authority: WO
Inventors: Soumitra Shankar Ghosh; Shahriar Mobashery
Original assignee: The Salk Institute Biotechnology/Industrial Associates, Inc.; Wayne State University
Priority date: 1989-10-13
Filing date: 1990-10-15
Publication date: 1991-05-02
Also published as: WO1991005555A3

Abstract

Inhibitors of the Zn+2-metalloendopeptidases, enkephalinase, angiotensin-converting enzyme, and collagenase, intermediates for synthesizing the inhibitors, and methods of making and using the inhibitors, are provided. The enkephalinase inhibitors of the invention are useful as analgesics or antihypertensives. The angiotensin-converting enzyme inhibitors of the invention are useful as anti-hypertensives. The collagenase inhibitors of the invention are useful in treating diseases, such as corneal ulceration, periodontal disease, and arthritis, which involve undesirable collagen degradation. The inhibitors of the invention are peptide or peptide ester derivatives of the intermediates of the invention, whichare N-acyl derivatives of amino acids of the formula X¿10?-NH-CHR9-(CO2H), wherein X10 is selected from the group consisting of N=C-CHR20-(C=O)- and X21(CR22R23)(CHR24)(C=O)-, wherein R20 is hydrogen or alkyl of 1 to 3 carbon atoms, R22, R23 and R24 are each independently hydrogen or alkyl of 1 to 3 carbon atoms, and X21 is fluoro, chloro or bromo; and R9 is benzyl, alkyl of 1-5 carbon atoms, or hydrogen.

Description

N-ACYL PEPTIDE METALLOENDOPEPTIDASE INHIBITORS AND METHODS OF USING SAME

TECHNICAL FIELD This invention relates generally to inhibitors of metalloendopeptidases and, more specifically, to inhibitors of enkephalinase, angiotensin-converting enzyme, and collagenase and therapeutic uses of the inhibitors and to intermediates for the synthesis of the inhibitors.

BACKGROUND OF THE INVENTION

Enkephalins, Met-enkephalin (Tyr-Gly-Gly-Phe-Met) and Leu-enkephalin (Tyr-Gly-Gly-Phe-Leu) , are pentapeptides which specifically bind opiate receptors in the brain and thereby are involved in regulation of nociceptive or pain stimuli. The enkephalins are generally short-lived molecules, being rapidly hydrolyzed into inactive fragments following their εynaptic release.

A variety of peptidases are known which are able to cleave enkephalins, in vitro, into biologically inactive fragments. Cleavage by an aminopeptidase results in release of the N-terminal tyrosine. A dipeptidylamino- peptidase has been implicated in the cleavage of the Gly²-Gly³ bond. The Zn⁺² metalloendopeptidases, enkephalinase (EC 3.4.24.11, also known as "neutral endopeptidase 24.11") (hereinafter referred to as "enkephalinase") and angiotensin-converting enzyme (EC 3.4.15.1, also known as "angiotensin I converting enzyme") (hereinafter referred to as "angiotensin- converting enzyme" or "ACE") cleave the Gly'-Phe⁴ bond.

It is widely accepted that enkephalinase is the enzyme primarily responsible for the in vivo hydrolytic cleavage of enkephalins and, as such, has a significant role in causing and regulating pain. Competitive inhibitors of enkephalinase are known which are active as antinociceptive agents (i.e., pain-relievers or "analgesics") in vivo in mammals, including humans. See, e.g., Erdos and Skidgel, FASEB J. 3, 145 (1989); Grazia et al., Eur. J. Pharmacol. 125, 147 (1986). Because enkephalinase is also known to proteolytically cleave, and thereby inactivate, the circulating form, ANF(99-126), of atrial natriuretic factor (ANF) , enkephalinase is thought to have a role in regulation of fluid balance and blood pressure. Indeed, enkephalinase inhibitors, by inhibiting the degradation of ANF(99-126) , might be employed in vivo to induce fluid and Na⁺ excretion and reduce blood pressure. Increases in urine volume and Na⁺ secretion are potentiated by, for example, the potent enkephalinase inhibitor thiorphan. See Erdos and Skidgel, supra.

Though, like enkephalinase, ACE cleaves enkephalin at the Gly-Phe bond, ACE's low affinity for enkephalins (^"1 mM) and relatively low rate of hydrolysis rule it out as a significant enzyme in the inactivation of endogenous enkephalins. ACE plays a significant role in blood pressure control, as the enzyme is primarily responsible for the conversion of the decapeptide angiotensin I, by proteolytic cleavage of the Phe⁸-His⁹ peptide bond, to the octapeptide angiotensin II, a potent vasoconstrictor. See, e.g., Erdos, Lab. Invest. 56, 345 (1987); Ondetti and Cushman, Ann. Rev. Biochem. 51, 283 (1982) ; Ehlers and Riordan, Biochemistry 28, 5311 (1989).

Competitive inhibitors of ACE, including captopril, enalaprilat and lisinopril, are used in vivo to reduce hypertension in humans.

Collagenases are Zn⁺² metalloendopeptidases involved in the turnover, remodeling or degradation of collagen and have been isolated from numerous species, from bacterial to human. The substrate specificities of collagenases vary, although they all proteolytically cleave a peptide bond in a collagen. The collagenases of Clostridium histolyticum (EC 3.4.24.3) catalyze cleavage of the X-Gly bond in the repeating sequence -Gly-Pro-X-Gly-Pro-X- of collagen, where X is frequently Ala or Hyp but may be any amino acid. The collagenase of Achromobacter iophagus (EC 3.4.24.8) catalyzes cleavage of the X-Gly bond in X-Gly-Pro-Y sequences. Lecroisey and Keil, Biochem. J. 179, 53 (1979). Mammalian collagenases have a recognition sequence of at least five amino acids and proteolytically cleave the Gly-Ile or Gly-Leu peptide bond in the sequence Pro-(Non-Pro),,-Gly-(lie or Leu)-(Non-Pro)₂. In most cases of mammalian collagenases that have been characterized, the amino acid on the carboxyl side of the scissile bond is lie, (Non-Pro)₁ is Leu or Gin, (Non-Pro)₂ is Ala, and there is an additional Gly at the amino terminal end and an additional Gly at the carboxy-terminal end of the pentapeptide, minimal recognition sequence. See Johnson et al., J. Enzyme Inhibition 2, 1 (1987).

Inhibitors of collagenase are thought to have a number of therapeutic applications, including treatment or inhibition of periodontal disease, via inhibition of both bacterial and human collagenases implicated in the disease; treatment or inhibition of collagen-degradative effects of bacterial infections, arising from bacterial collagenase activity; treatment of corneal ulceration that is caused, at least in part, by collagenase-catalyzed collagen degradation; treatment of arthritis, including rheumatoid arthritis and osteoarthritis; and inhibition or prevention of tumor metastasis. See Johnson et al., supra.

A number of competitive inhibitors of bacterial and mammalian collagenases are known. See Johnson et al., supra; Vencill et al., Biochemistry 24, 3149 (1985); Yiotakis et al., Eur. J. Biochem. 172, 761 (1988); Galardy and Grobelny, Biochemistry 22, 4556 (1983); Grobelny and Galardy, Biochemistry 24, 6145 (1985) ; Mookhtiar et al. (II), Biochemistry 27, 4299 (1988); Gray et al., Biochem. Biophys. Res. Comm. 101, 1251 (1981); Clark et al.. Life Sciences 37, 575 (1985); Wallace et al., Biochem. J. 239, 797 (1986); and Mookhtiar et al. (I), Biochemistry 26, 1962 (1987) . Zn⁺-metallopeptidase inhibitors, including those of the present invention, to be described in detail below, may also find antibacterial application against bacteria whose pathogenicity depends at least in part on Zn⁺²-metallo- peptidases produced by the bacteria.

Information about Zn⁺² metallopeptidases gained from a variety of different types of studies has provided a basis for the design of inhibitors of the enzymes. Thus, numerous chemical and kinetic studies of synthetic substrates and inhibitors of the various enzymes have led to suppositions about the three-dimensional structures that an inhibitor would need to have for a good fit in the active site of an enzyme and about the chemical inter¬ actions involved in catalysis of proteolysis by an enzyme or inhibition of such catalysis. The availability of high-resolution molecular structures from X-ray diffraction studies of crystallized the Zn⁺² metallopeptidases carboxypeptidase A and thermolysin, coupled with evidence that the Zn⁺²-containing active sites of Zn⁺²-metallopep- tidases are similar in molecular structure and function chemically in similar ways in their catalytic activities, has provided additional information to guide the design of inhibitors that have appropriate structural and chemical properties to be inhibitors that are specific for one or a few of the types of Zn⁺² metalloendopeptidases. The availability of amino acid sequences for metalloendopep¬ tidases, including carboxypeptidases A, thermolysins from various sources, enkephalinases from various sources, and ACE's from various sources, has provided additional information suggestive of structures of active sites and binding sites for substrates and inhibitors and the structural requirements and chemical attributes of desirable inhibitors.

Still, the art of predicting compounds that will be effective as inhibitors of a particular Zn⁺² etalloendo- peptidase (i.e., enkephalinase, ACE, or collagenase) and designing inhibitors based on such predictions remains uncertain. (To be regarded as effective as an inhibitor, in the case of a competitive inhibitor, a compound should have a K_? of less than about 50 μM.) Experience has shown that, notwithstanding information that might be available on an enzyme from studies of its molecular structure, its primary sequence, and the physical and chemical properties of its substrates and inhibitors, there remain numerous ill-understood factors that affect whether a particular compound will be an effective inhibitor. Predicting compounds that will be specific inhibitors for a particular type of Zn⁺² metalloendopeptidase (i.e., enkephalinase or ACE or collagenase, with, in the case of a competitive inhibitor, an inhibition constant, K _r for one type that is at least about two orders of magnitude lower than that for the other types) is even more uncertain, because often subtle, ill-understood differences among the enzymes are important in such predictions. Still more uncertain is the design of so-called "mechanism-based" inhibitors for enzymes, including Zn⁺² metalloendo-peptidases, as such inhibitors must not only, like competitive inhibitors, physically occupy the active site of an enzyme to block access thereto of substrate but also be positioned with sufficient precision and stability in the active site to undergo chemical reaction(s) there to unmask reactivity of a functional group so that the activated functional group, in turn, can form a covalent bond with a moiety of the enzyme, usually in or near the active site. The task of designing inhibitors for an enzyme is further complicated when, as with enkephalinase, ACE and collagenase, the three-dimensional structure of the enzyme to atomic resolution (as from X-ray crystallographic studies) , which can reveal many of the details pertinent for rational design of inhibitors of the enzyme, is not available to guide the design.

The known inhibitors for enkephalinase, ACE and collagenases are competitive inhibitors. As such, the inhibitors are only transiently held, non-covalently, in the enzyme's active site and are effective in blocking peptidase activity on natural substrates only during the time that they occupy the active site of the enzyme in a way that blocks access thereto in a reactive orientation of such a substrate. Dissociation of the enzyme-inhibitor complex frees the enzyme to act upon its natural substrate. Undesirably, as competitive enzyme inhibitors are degraded or otherwise cleared from the body, the activity of the enzyme intended to be inhibited is quickly and substan¬ tially fully restored, because no enzyme is irreversibly inactivated by competitive inhibitors. Nonetheless, it would be desirable to have additional competitive inhibitors for enkephalinase, ACE and collagenases, particularly ones that have low inhibition constants (below about 1 Nm) for at least one of the enzymes of a species (especially human) or that are specific for one of the three types of enzymes (i.e., an inhibitor that has an inhibition constant for one of the types of enzyme that is in the nanomolar range and an inhibition constant for the other types of enzyme of the same species that is at least about 100 to 1000 times greater) .

It would also be desirable to have irreversible inhibitors of enkephalinase, ACE and collagenases, which, by permanently inactivating the enzymes, would provide longer-lived inhibition thereof. In particular, mechanism-based inhibitors for the enzymes would be especially desirable. A mechanism-based inhibitor, otherwise sometimes referred to as a "suicide inhibitor," is capable, once it has formed a Michaelis complex through non-covalent interactions in the active site of the enzyme to be inhibited, of chemically interacting with moieties of the enzyme in the active site in a manner which enables a "latent" functional group of the inhibitor to be activated (sometimes referred to as "unmasked") so that the inhibitor then reacts, and forms covalent bonds, with residue(s) of the enzyme in, or very close to, the active site. If, in an encounter of the inhibitor with an active site of the enzyme to be inhibited, stable covalent bonds with the enzyme are formed, the enzyme will be irreversibly inhibited, because the active site will be permanently occupied or blocked by the inhibitor. See, e.g., Walsh, Ann. Rev. Biochem. 53, 493 - 535 (1984) ; Walsh, Tetrahedron Lett. 38, 871 - 908 (1982).

Particularly preferred would be a mechanism-based inhibitor which would irreversibly inactivate only one type of Zn⁺² etallopeptidase (e.g., only enkephalinase, or only ACE, or only collagenase) of a mammalian, and particularly the human, species.

SUMMARY OF THE INVENTION

The invention entails potent inhibitors of enkephalinase, ACE and collagenases, which are peptide derivatives of the N-(cyanoacetyl) and N-(3-halopropionyl) derivatives of amino acids. We have discovered surprisingly that these N-acyl derivatives of L-phenylalanine are mechanism-based inhibitors of carboxypeptidase A rather than merely substrates for proteolytic cleavage by the enzyme at the peptide bond. The partition ratios for inactivation (relative to peptide bond hydrolysis) of N-(cyanoacetyl)-L- phenylalanine and N-(3-chloropropionyl)-L-phenylalanine with carboxypeptidase A are about 1200 and about 1700, respectively.

The N-acyl derivatives of amino acids, particularly L- phenylalanine, glycine, L-alanine, L-leucine, L-valine, and L-isoleucine, are part of the invention. They are useful as intermediates for making the inhibitors of enkephalinase, ACE and collagenase of the invention by straightforward chemistry well known to the skilled. The enkephalinase, ACE and collagenase inhibitors of the invention are compounds of Formula I

X^NH-CHR,-(C=0)-X₂

I, wherein X-, is a functional group from which a Zn⁺² metalloendopeptidase, at its active site, is capable of abstracting a proton to yield an activated functional group capable of forming a stable, covalent bond with a residue in the active site; Ε^ is benzyl, alkyl of 1 to 5 carbon atoms, or hydrogen; X₂ is joined to the -CO- in an amide linkage and is selected from the group consisting of glycine, N-methyl-glycine, N-benzyl-glycine, D-alanine, L-alanine, 0-alanine, D-phenylalanine, L-phenylalanine, D-leucine, L-leucine, 3-amino propionic acid, D-proline, L-proline, and the group X₃-X₄, wherein X₃ is joined to the -CHR^CO- group in an amide linkage and is selected from the group consisting of L-proline, L-alanine, L-valine,

L-leucine, and L-0-methy1tyrosine; when X₃ is L-proline or L-alanine, X₄ is selected from the group consisting of L-arginine, L-proline, L-leucine, L-alanine, L-hydroxyproline, and L-homoarginine; and when X₃ is L- valine, L-leucine, or L-O-methyltyrosine, X₄ is selected from the group consisting of glycine, L-alanine, and the alkyl esters of glycine and alanine, wherein the alkyl is of 1 - 5 carbons; provided that if 1^ is ethyl or benzyl, X₂ is not X₃-X₄; if X₂ is X₃-X₄ and X₃ is L-proline or L-alanine, R_ς is hydrogen or methyl; and if X₂ is X₃-X₄ and X₃ is L- leucine, L-valine or L-O-methyltyrosine, Εi^ is alkyl of 3 - 5 carbon atoms; and physiologically acceptable salts thereof.

It is intended that all stereoisomers be included in the compounds of Formula I.

Among the groups X, are a group of formula (N≡C) (CHR₁₀) (C=0)-, wherein R₁₀ is hydrogen or alkyl of 1 to 3 carbons and with which isomerization can occur by proton abstraction, with transfer of a proton from the α-carbon, to give rise to a reactive α-keto-ketenimine; a group of formula X₅(CR₁₁R₁₂) (CHR₁₃) (C=0)-, wherein X₅ is a good leaving group, such as fluoro, chloro or bromo, R , R₁₂ and R₁₃ are independently selected from hydrogen or alkyl of 1 to 3 carbon atoms, and with which proton abstraction can occur in an elimination reaction, with loss of a hydrogen from the carbon alpha to the carbonyl group and the X₅ group from the carbon beta to the carbonyl group, leading to formation of an α, 0-unsaturated amide, a reactive Michael acceptor; and a group of formula HC≡C(CHR_U) (C=0)-, wherein R is hydrogen or alkyl of 1 to 3 carbon atoms, with which proton abstraction can lead to an allenic amide, also a reactive Michael acceptor.

The inhibitors of the invention are useful as analgesics (i.e., the enkephalinase inhibitors) or antihypertensives (i.e., the ACE inhibitors or the enkephalinase inhibitors) ; and in antibacterial or therapeutic applications involving collagenase inhibition. The invention encompasses methods of treating pain or hypertension in mammals, including humans, suffering therefrom by administering to such a mammal an effective amount of an analgesic or antihypertensive, respectively, according to the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compounds of Formula I, described supra. The compounds of Formula I are inhibitors of enkephalinase, ACE, bacterial collagenase or mammalian collagenase.

The present invention also encompasses compounds of Formula II

X₁-NH-CHR₉-(C0₂H)

II, wherein X₁ and R_γ are as defined for Formula I, and salts thereof. The compounds of Formula II are intermediates for the synthesis of the compounds of Formula I by straightforward methods well known in the art. Surprisingly, the compounds of Formula II, wherein ^~^ is benzyl and the configuration at the carbon to which the benzyl group is bound is R (i.e., the compounds of Formula II which are N-acyl analogs of L-phenylalanine) have been discovered to be mechanism-based inactivators of carboxypeptidase A. The invention also encompasses the method of making a compound of Formula I, wherein X₂ is an amino acid or dipeptide, which comprises using mixed anhydride to couple the compound of Formula II, with the same substituents for X., and R, as the compound of Formula I, with the t-butyl ester of the amino acid or dipeptide corresponding to the group X₂ in the compound of Formula I, and then hydrolyzing the resulting t-butyl ester of the compound of Formula I by acidic hydrolysis. The invention further includes the method of making a compound of Formula I, wherein X₂ is a dipeptide alkyl ester, which comprises using mixed anhydride to couple the compound of Formula II, with the same substituents for X₁ and R, as the compound of Formula I, with the alkyl ester of the dipeptide corresponding to the group X₂ in the compound of Formula I.

It is preferred that the configuration at the carbon to which ^ is bound in the compound of Formula II be R (i.e., that the corresponding amino acid be L) , if R_ς is other than hydrogen. Similarly, it is preferred that the amino acid(s), other than glycine, in the group X₂ be L- amino acids. If desired, with compounds of Formula I which have diastereomers, the diastereomers can be separated by reverse phase chromatography. The mixed anhydride coupling of the method of making a compound of Formula I in accordance with the invention is readily carried out at room temperature by adding sequentially to the compound of Formula II in a suitable solvent, such as chloroform or tetrahydrofuran ("THF") under N₂, N-methyl morpholine and isobutylchloroformate, followed by adding the alkyl ester of the dipeptide corresponding to X₂, if the compound of Formula I is an ester, or the t-butyl ester of the amino acid or dipeptide corresponding to X₂, if the compound of Formula I is not an ester, along with additional N-methyl morpholine and letting the reaction proceed for at least several hours. Finally, the reaction mixture can be diluted with a large excess of ether, followed by washing with 5 % citric acid, saturated sodium bicarbonate and brine. The ethereal layer can then be dried over anhydrous magnesium sulfate and concentrated in vacuo to yield the compound of Formula I, if an alkyl ester, or the t-butyl ester of the compound of Formula I.

If the compound of Formula I is not an ester, the compound of Formula I can then be obtained, in accordance with the method of the invention, by a standard acidic hydrolysis of the t-butyl ester. Thus, for example, the t-butyl ester is taken up in a 30 % - 50 % solution of trifluoroacetic acid (TFA) in methylene chloride and reaction is allowed to proceed at room temperature for 30 - 60 minutes. Then the solution is evaporated to dryness and the residue is taken up in ethylacetate, which is washed with saturated NaHS0₄ and then with brine. The washed solution is finally dried over anhydrous magnesium sulfate, and finally evaporated in vacuo to yield the compound of Formula I. If desired, the compound of Formula I can be further purified by any standard method available in the art, such as HPLC or reverse phase chromatography (which, as noted above, can also be employed to separate diastereomers, if desired) . See also examples 1 and 2 for further details on mixed anhydride coupling.

One discovery which underlies the present invention is that the presence of a functional group X, in a compound which is capable of binding in the active site of a Zn⁺² metalloendopeptidase provides a mechanism-based inhibitor for the enzyme.

Another discovery underlying the invention is that the peptide bond between X, and -(CHR₉)-X₂ in a compound of Formula I is not simply hydrolyzed by a Zn⁺² metalloendopeptidase but rather the compound, through activation of X., in the active site, does inactivate the enzy e with a significant partition ratio (of inactivation relative to peptide bond hydrolysis) .

Preferred inhibitors in accordance with the invention are those wherein X, is (NC) (CH₂) (C=0)- or C1(CH₂)₂(C=0)-.

In addition to the compounds of the invention, the invention provides a method of reducing pain in a mammal suffering therefrom comprising administering to said mammal a pain-reducing-effective amount of a compound of Formula XII:

X_rNH-CH(CH₂-(θ))-(C=0)-x₁₂

XII, wherein X₁₂ is joined to the -(C=0)- in an amide linkage and is selected from the group consisting of glycine, N-benzyl-glycine, L-alanine, D-alanine, L-phenylalanine, D-phenylalanine, L-leucine, D-leucine, and 3-amino propionic acid; or a pharmacologically acceptable salt thereof.

Still further, the invention provides a method for treating hypertension in a mammal suffering therefrom comprising administering to said mammal an antihyper- tensive-effective amount of a compound of Formula I, wherein X₂ is other than a dipeptide; or a physiologically acceptable salt thereof. Still further, the invention provides a method for inhibiting collagenase comprising combining with a collagenase a collagenase-inhibiting effective amount of a compound of Formula I, wherein X₂ is a dipeptide or a dipeptide ester; or a physiologically acceptable salt thereof. Therapeutic applications of collagenase inhibition are cited supra.

Reference herein to a compound or a formula for a compound is, unless otherwise qualified, to all stereoisomers of the compound. The designation of "R" or "S" as the configuration at an asymmetric carbon of a compound is based on Cahn-Ingold-Prelog convention rules. Reference to an amino acid, unless the configuration at its asymmetric carbon is specified, is to the L-enantiomer. Three letter abbreviations used for amino acids are the standard three letter abbreviations used in the art, including "Har" for L-homoarginine and "Hyp" for 4-hydroxy- L-proline (L-hydroxyproline) .

The compounds of Formula I of the present invention are inhibitors of enkephalinase or angiotensin-converting enzyme ("ACE") or both of these enzymes from mammals, including humans, or inhibitors of bacterial or mammalian (or both bacterial and mammalian) collagenases.

The amino acid sequences of the human, rat and rabbit enkephalinases have been deduced from cDNAs for the enzymes. Malfroy et al., Biochem Biophys. Res. Comm. 144, 59 - 66 (1987); Devault et al., EMBO J. 6, 1317 - 1322

(1987); Malfroy et al., FEBS Lett. 229, 206 - 210 (1988). The amino acid sequences of human ACE is provided by Soubrier et al., Proc. Natl. Acad. Sci. (USA) 85, 9386 (1988) . Methods for preparing physiologically acceptable salts of the compounds of Formula I, which are weak acids, are well known. Among such salts are the sodium, potassium, ammonium, magnesium, and calcium salts.

Especially preferred among the endopeptidase inhibitors of the invention, of Formula I, are those which are mechanism-based inhibitors of enkephalinase or ACE or both.

The compounds of Formula I, which are mechanism-based inhibitors, are also necessarily substrates of the enzyme. In some encounters between such a compound of the invention and enkephalinase, ACE, or a collagenase, the compound will be changed (activated by a reaction involving proton abstraction from X,) in a reaction catalyzed by the enzyme and will diffuse away from the active site of the enzyme before a covalent bond with a moiety in the active site can be formed. In other encounters, hydrolysis of the peptide bond between X₁ and -(Cffi^)- will occur and preclude activation of X, that can result in inactivation of the enzyme. In still other encounters, however, after the reaction to "activate" X., occurs, a subsequent reaction with a moiety in the active site of the enzyme will occur with the activated inhibitor to effect a covalent linkage between the inhibitor and the enzyme and irreversibly inactivate the enzyme. The "partition ratio" of inactivation of an enzyme by a mechanism-based enzyme inhibitor with an enzyme is defined as the negative of the time derivative of the concentration of the inhibitor divided by the time derivative of the concentration of inactivated enzyme. The partition ratio is one less than the average number of molecules of inhibitor with which the enzyme must catalyze either hydrolysis of the X, - (CHR^ peptide bond or formation of an activated intermediate before an activated intermediate will react with and inactivate the enzyme. Measurement of the partition ratio of inactivation of an enzyme by a mechanism-based inhibitor is readily carried out by the skilled. A partition ratio for inactivation of an enzyme by a mechanism-based inhibitor of 5000 or less is desirable; especially preferred are partition ratios of 2000 or less. A measure of the specificity of a mechanism- based inhibitor for one of a set of enzymes is provided by the partition ratios for inactivation of the various enzymes by the inhibitor; if the partition ratio for one of the enzymes is very much lower than those for the other enzymes, the mechanism-based inhibitor can be said to be specific in mechanism-based inhibition for the one enzyme of the set. The effectiveness of the enkephalinase inhibitors, ACE inhibitors and collagenase inhibitors of the invention as antinociceptive, antihypertensive and collagenase inhibiting agents, respectively, is ascertained by their ability to inhibit purified enkephalinase, ACE or collagenase, respectively, in vitro.

The compounds listed in Tables 1, 2, 3 and 4 are particularly preferred enkephalinase, ACE and collagenase inhibitors, respectively. It will be noted that several compounds are both ACE inhibitors and enkephalinase inhibitors.

TABLE 1

ENKEPHALINASE INHIBITORS^*

NC-CH₂-CO-NH-CH (CH₂C₆H₅) -CO-L-glycine

NC-CH₂-CO-NH-CH (CH₂C₆H₅) -CO-L-alanine

NC-CH₂-CO-NH-CH (CH₂C₆H₅) -CO-D-alanine NC-CH₂-CO-NH-CH ( CH₂C₆H₅ ) -CO-NH-CH₂-CH₂-COOH

NC-CH₂-CO-NH-CH (CH₂C₆H₅) -CO-L-leucine

NC-CH₂-CO-NH-CH (CH₂C₆H₅) -CO-L-phenylalanine

Cl (CH₂) ₂-CO-NH-CH (CH₂C₆H₅) -CO-L-glycine

Cl (CH₂) ₂-CO-NH-CH (CH₂C₆H₅) -CO-L-alanine Cl (CH₂) ₂-CO-NH-CH (CH₂C₆H₅) -CO-D-alanine

Cl (CH₂) ₂-CO-NH-CH (CH₂C₆H₅) -CO-NH-CH₂-CH₂-COOH

Cl (CH₂) ₂-CO-NH-CH (CH₂C₆H₅) -CO-L-leucine

Cl(CH₂)₂-CO-NH-CH(CH₂C₆H₅)-CO-L-phenylalanine

^*Preferred configuration of the phenylalanyl residue is L.

TABLE 2 ACE INHIBITORS^*

NC-CH₂-CO-NH-CH (CH₃) -CO-L-proline

NC-CH₂-CO-NH-CH₂-CO-L-proline NC-CH₂-CO-NH-CH ( CH₂C₆H₅ ) -CO-L-prol ine

NC-CH₂-CO-NH-CH (CH₂C₆H₅) -CO-L-alanine

NC-CH₂-CO-NH-CH (CH₂C₆H₅) -CO-D-alanine

NC-CH₂-CO-NH-CH (CH₂C₆H₅) -CO-L-phenylalanine

NC-CH₂-CO-NH-CH(CH₂C₆H₅) -CO-L-leucine NC-CH₂-CO-NH-CH (CH₂C₆H₅) -CO-L-glycine

NC-CH₂-CO-NH-CH (CH₂C₆H₅) -CO-N (CH₃) -CH₂-COOH

Cl (CH₂) 2-CO-NH-CH (CH₃) -CO-L-proline

Cl (CH₂) ₂-CO-NH-CH₂-CO-L-proline

Cl (CH₂) 2-CO-NH-CH (CH₂C₆H₅) -CO-L-proline Cl (CH₂) ₂-CO-NH-CH (CH₂C₆H₅) -CO-L-alanine

Cl (CH₂) ₂-CO-NH-CH (CH₂C₆H₅) -CO-D-alanine

Cl(CH₂)₂-CO-NH-CH(CH₂C₆H₅)-CO-L-phenylalanine

Cl(CH₂)₂-CO-NH-CH(CH₂C₆H₅)-CO-L-leucine

Cl (CH₂) ₂-CO-NH-CH (CH₂C₆H₅) -CO-L-glycine Cl ( CH₂ ) ₂-CO-NH-CH ( CH₂C₆H₅ ) -CO-N ( CH₃ ) -CH₂-COOH

^*Preferred configuration of the phenylalanyl and alanyl residues is L.

TABLE 3 BACTERIAL COLLAGENASE INHIBITORS NC-CH₂-CO-NH-CH₂-CO-L-proline-L-arginine NC-CH₂-CO-NH-CH₂-CO-L-proline-L-proline NC-CH₂-CO-NH-CH₂-CO-L-proline-L-leucine NC-CH₂-CO-NH-CH₂-CO-L-proline-L-alanine NC-CH₂-CO-NH-CH₂-CO-L-proline-L-hydroxyproline NC-CH₂-CO-NH-CH₂-CO-L-proline-L-homoarginine NC-CH₂-CO-NH-CH₂-CO-L-alanine-L-arginine NC-CH₂-CO-NH-CH₂-CO-L-alanine-L-proline NC-CH₂-CO-NH-CH₂-CO-L-alanine-L-leucine NC-CH₂-CO-NH-CH₂-CO-L-alanine-L-alanine

NC-CH₂- CO-NH-CH₂-CO-L-alanine-L-hydroxyproline NC-CH,- CO-NH-CH₂-CO-L-alanine-L-homoarginine

₂-CO-NH-CH₂-CO-L-proline-L-arginine

₂-CO-NH-CH₂-CO-L-proline-L-proline

₂-CO-NH-CH₂-CO-L-proline-L-leucine

₂-CO-NH-CH₂-CO-L-proline-L-alanine

₂-CO-NH-CH₂-CO-L-proline-L-hydroxyproline

₂-CO-NH-CH₂-CO-L-proline-L-homoarginine

₂-CO-NH-CH₂-CO-L-alanine-L-arginine

₂-CO-NH-CH₂-CO-L-alanine-L-proline

₂-CO-NH-CH₂-CO-L-alanine-L-leucine

₂-CO-NH-CH₂-CO-L-alanine-L-alanine

₂-CO-NH-CH₂-CO-L-alanine-L-hydroxyproline

₂-CO-NH-CH₂-CO-L-alanine-L-homoarginine

TABLE 4

MAMMALIAN COLLAGENASE INHIBITORS^*

NCCH₂-CO-NH-CH('Bu)-CO-L-leucine-glycine ethyl ester

NCCH_g-CO-NH-CHC^'BuJ-CO-L-leucine-L-alanine ethyl ester NCCH^CO-NH-CH BuJ -CO-L-O-methyltyrosine-glycine ethyl ester

NCCH₂-CO-NH-CH(*Bu)-CO-L-O-methyltyrosine-L-alanine ethyl ester

NCCΕ^-CO-NH-CHfBuJ-CO-L-valine-glycine ethyl ester NCCH₂-CO-NH-CH(⁵Bu)-CO-L-valine-L-alanine ethyl ester Cl(CH₂)₂-CO-NH-CH(*Bu)-CO-L-leucine-glycine ethyl ester Cl(CH₂)₂-CO-NH-CH('Bu)-CO-L-leucine-L-alanine ethyl ester

Cl(CH₂)₂-CO-NH-CH('Bu)-CO-L-O-methyltyrosine-glycine ethyl ester Cl(CH₂)₂-CO-NH-CH('Bu)-CO-L-O-methyltyrosine-L-alanine ethyl ester

C1(CH₂)₂-CO-NH-CH( Bu)-CO-L-valine-glycine ethyl ester

Cl(CH₂)₂-CO-NH-CH('Bu)-CO-L-valine-L-alanine ethyl ester 'Bu = isobutyl. ^*Preferred configuration at the carbon bound to the 'Bu group is R.

Methods of assaying enkephalinase inhibitors, ACE inhibitors, and collagenase inhibitors for capacity to inhibit the respective enzymes, including methods of purifying enzymes for use in the assay methods, are known to those skilled in the study of the enzymes. Methods for enkephalinase inhibitors, ACE inhibitors and bacterial collagenase inhibitors are described in the Examples. For mammalian collagenase inhibitors, see Johnson et al., supra, at pages 4 - 5, and the references cited there.

It is contemplated that the inhibitors of the invention will be administered under the guidance of a physician or veterinarian to relieve pain in a human or other mammal suffering therefrom (in the case of the enkephalinase inhibitors of the invention) or to reduce blood pressure in a human or other mammal suffering from hypertension (in the case of the ACE inhibitors or enkephalinase inhibitors of the invention) , or for both purposes in the case of inhibitors of the invention which are effective as inhibitors of both enkephalinase and ACE, or for any of a number of therapeutic applications, such as treatment of corneal ulcers or periodontal disease, in the case of inhibitors of the invention which are effective as inhibitors of collagenases.

With respect to the enkephalinase or ACE inhibitors, administration will be parenterally, preferably intravenously, in unit doses or by continuous infusion, of an inhibitor or a physiologically acceptable salt thereof dissolved in any physiologically acceptable diluent, such as physiological saline, phosphate buffered saline, or the like. The route of administration (e.g., intravenous, intramuscular, intraperitoneal, subcutaneous) , mode of administration (e.g., by unit doses or continuous infusion) , and dosage regimen will vary somewhat depending on the inhibitor employed, the species, age, weight and general medical condition of the mammal being treated, and the particular condition of the mammal for which the inhibitor is being administered. Determining these factors for a particular mammal being treated for a particular condition with a particular inhibitor will be routine for the pharmacologist, physician or veterinarian of ordinary skill. Generally, in the case of treating humans with an inhibitor according to the invention, a dose of inhibitor or physiologically acceptable salt thereof of between about 0.01 mg/kg body weight per day and 100 mg/kg body weight per day, infused continuously, administered in several equal doses per day, or administered in a single dose per day, will be effective to relieve pain (in the case of enkephalinase inhibitors) or reduce hypertension (in the case of ACE inhibitors or enkephalinase inhibitors) .

With respect to the collagenase inhibitors according to the invention, administration may be topical in a suitable, physiologically acceptable vehicle (e.g., cream, solution) for application to the eye, in the case of use for treatment of corneal ulceration, or application into the gingival crevice or subgingival space, in the case of use for treatment of periodontal disease. The collagenase inhibitors may also be administered parenterally, in unit doses or by continuous infusion, at or near the site on the body of the mammal being treated at which inhibition of collagen degradation is desired. The inhibitor or a physiologically acceptable salt thereof will, for administration, be dissolved in any physiologically acceptable diluent, such as physiological saline, phosphate buffered saline, or the like. The route of administration, mode of administration (e.g., by unit doses or continuous infusion) , and dosage regimen will vary somewhat depending on the inhibitor employed, the species, age, weight and general medical condition of the mammal being treated, and the particular condition of the mammal for which the inhibitor is being administered. Determining these factors for a particular mammal being treated for a particular condition with a particular inhibitor will be routine for the pharmacologist, physician or veterinarian of ordinary skill. Generally, in the case of treating humans with a collagenase inhibitor according to the invention, a dose of inhibitor or physiologically acceptable salt thereof of between about 0.1 mg per day and 100 mg per day, infused continuously, or administered by any route, including topically or by injection into or near the site at which collagenase inhibition is desired, in several equal doses per day, or a single dose per day, will be effective to achieve the desired inhibition of collagen degradation. In certain applications, such as treating periodontal disease, inhibitors according to the invention of bacterial collagenases and mammalian collagenases may be employed in combination.

The following Examples are included to aid in the understanding of the invention. They are not intended to limit the scope of the invention as defined by the appended claims. EXAMPLE 1 N-(Cyanoacetyl)-L-phenylalanine methyl ester

To a solution of 645 mg (3 mmol) of L-phenylalanine methyl ester-HCl in 12 L of 1:1 THF/DMF, N-methylmorpholine (0.327 mL, 3 mmol) was added under an atmosphere of argon, and the mixture was stirred for 5 in. Cyanoacetic acid (255 g, 3 mmol) and l-ethyl-3-(3-dimethylaminopropyl)- carbodiimide-HCl (573 mg, 3 mmol) were then added and the mixture was stirred for 16 hours. The reaction mixture was subsequently concentrated in vacuo, taken up in ethyl acetate (100 mL) , washed with water and brine (30 mL each), followed by the drying of the organic solution over anhydrous MgS0₄. The solution was concentrated to provide 550 mg of the title compound as a white solid. Yield 61%; mp 110-lll^βC; IR (CHCl₃) : 3302, 2943, 2259, 1738, 1651 cm^"1; ¹H NMR (CDC1₃) : delta 7.30 (m, 3H) , 7.06 (m, 2H) , 6.40 (br s, 1H) , 4.86 (m, 1H) , 3.76 (s, 3H) , 3.35 (d, 2H) , 3.16 (m,2H); El MS m/z 246.1002 (2%, M) (expected 246.1004).

N-(Cyanoacetyl)-L-phenylalanine

A suspension of (N-cyanoacetyl)-L-phenylalanine methyl ester (550 mg, 2.24 mmol) in water (20 L) was allowed to react with a solution of 80 mg of cβ-chymotrypsin (EC 3.4.21.1) in 5 L of water. A constant pH of 7.2 was maintained by titrating the mixture with 0.05 M NaOH using a pH-stat apparatus. After 15 hrs. "40 mL of the NaOH solution was consumed, indicating that the hydrolysis was essentially complete. The aqueous solution was washed with ether, and then was acidified to pH 2.0 with 1 N HC1. The solution was concentrated to dryness in vacuo. The residue was sonicated in the presence of 100 mL ethyl acetate, and the resultant solution was filtered and dried over anhydrous MgS0₄. Evaporation of the organic solvent furnished 437 mg of the product as a white solid. Yield, 85%; mp 156-158°C; analytical HPLC single peak, t_R = 11.2 min. (Vydac, Ultrasphere ODS, 0.46 X 25 cm, 5-95% linear acetonitrile gradient in 0.1 % aqueous TFA over 30 min., 1 mL/min, 220 nm) ; IR (CHC1₃) : 3317, 3066, 2926, 2266, 1729, 1672 cm^"1; ¹H NMR (CDCl₃) : delta 7.67 (br d 1H, J = 6.9 Hz), 7.26 (m, 5H) , 4.75 ( , 1H) , 3.62 (s, 2H) , 3.22 (dd, 1H, J = 5.3 and 13.9 Hz), 3.06 (dd, 1H, J - 7.5 and 13.9 Hz); El MS m/z 232.0850 (1%, M) (expected 232.0847).

EXAMPLE 2 N-(3-Chloropropionyl)-L-phenylalanine methyl ester

N-methylmorpholine (0.22 mL, 2 mmol) was added to a suspension of L-phenylalanine methyl esterΗCl (439 mg, 2 mmol) in 10 mL dry THF under an atmosphere of argon. The mixture was stirred for 10 min. , followed by the dropwise addition of 3-chloropropionyl chloride (0.192 mL, 2 mmol). Subsequently, an additional portion of N-methylmorpholine (0.22 mL, 2 mmol) was added and the mixture was stirred for 16 hrs. The solution was taken up in ethyl acetate (100 L) and was washed successively with saturated NaHC0₃ (30 L) and brine (30 mL) . The organic layer was dried over MgS0₄ and was concentrated to dryness in vacuo to afford 500 mg of the product as a white solid. Yield, 93%; mp

62-64^βC; IR (CDC1₃) : 3299, 1745, 1652, 1737 cm^"1; -^~ NMR (CDC1₃) : delta 7.3-7.1 (m, 5H) , 6.07 (br d, 1H) , 4.93 (m, 1H) , 3.77 (m, 2H) , 3.75 (s, 3H) , 3.15 ( , 2H) , 2.65 (m, 2H) ; El MS m/z 269.0821 (1%, M) (expected 269.0818).

N-(3-Chloropropionyl-L-Phenylalanine. α-chymotrypsin catalyzed hydrolysis of 250 mg (0.93 mmol) of the methyl ester, according to the methodology described for the preparation of N-(cyanoacetyl)- L-phenylalanine, provided 200 mg of the title compound as a white solid. Yield, 84%; mp 122-124^βC; analytical HPLC single peak, t_R = 15.0 min (conditions as described for N-(cyanoacetyl)-L-phenylalanine) . ¹H NMR (CDC1₃) : delta 7.3-7.1 (m, 5H) , 6.14 (d, 1H, J - 7.4 Hz) , 4.95 (m, 1H) , 3.76 (m, 2H) , 3.20 (m, 2H) , 2.66 (m, 2H) ; El MS m/z 255.0664 (2%, M) (expected 255.0662) . EXAMPLE 3 Enkepalinase from rabbit kidney cortex was purified by immunoaffinity chromatography using a monoclonal antibody. Biochem. Biophys. Res. Commun. 131, 255 (1985) . The assay for enkephalinase is carried out in vitro using the fluorescent substrate, dansyl-D-Ala-Gly-Phe-(p-N0₂)-Gly (referred to herein as "DAGNPG") in accordance with the procedure of Florentin et al., Anal Biochem. 141, 62 (1984). Fluorometric assays are performed at 37^βC on a spectrofluorometer (e.g. Gilford Fluoro IV, Gilford Instruments Co.)equipped with a temperature-controlled cell holder.

The enkephalinase inactivator assay is carried out as follows: Enkephalinase (4 μg) and various amounts of inhibitor are mixed (final volume = 53μl) and inactivation is allowed to occur at 23*C for 5 hours. Reaction mixtures contain 0.15 M Tris acetate, 1% octylglucoside, 5% dioxane, pH 7.4, 3.8 μg enkephalinase (rabbit kidney) and varying amount of enkephalinase inhibitor. At various times, a 5 μl aliquot of the reaction mixture is withdrawn and added to an assay solution which contains 0.1 mM DAGNPG and 50mM Tris hydrochloride, pH 7.4. Initial reaction rates (for the first 10% of the reaction) are monitored continuously by measuring the increase of fluorescence at an excitation wavelength of 342 nm and emission wavelength of 562 nm. A plot of the initial rate versus time gives a time course of inactivation of the enzyme.

EXAMPLE 4 Angiotensin converting enzyme from frozen rabbit lung is purified according to the procedure of Das and Soffer, J. Biol. Chem. 250, 6762 (1975).

The enzyme is assayed using hippuryl-L-histidyl- L-leucine according to the procedure of Cheung and Ondetti, Biochim. Biophys. Acta 293, 451 (1973), by following the release of histidyl-leucine in 100 mM potassium phosphate, 300 mM NaCl, pH 8.3 and a single 30 minute time point. The reaction is initiated by the addition of enzyme and terminated by addition of 0.3 M NaOH. The fluorometric assay of the released histidyl-leucine is then performed using o-pthaldialdehyde in methanol, followed by addition of 3 M HCl, and measuring the fluorescence at 500 nm, using excitation at 365 nm.

The inactivation assay is carried out as follows: ACE and various amounts of inhibitor in 50 mM potassium phosphate, 100 mM NaCl, pH 8.3 are incubated in a total volume of 100 μl. At various time points, a 5 μl aliquot of the reaction mixture is withdrawn and assayed for enzyme activity according to the procedure described above.

EXAMPLE 5 Collagenase A obtained from Sigma Chemical Co.

(Catalog No. C0773, Type VII, 95 % protein) is employed in assay and inactivation studies.

2-furanacryloyl-L-leucylglycyl-L-prolyl-L- alanine (referred to herein as "FALGPA") is used for the assay of the synthetic substrate. The concentration was 0.05 mM in 50 mM tricine, 0.4 M NaCl, 10 mM CaCl₂, pH 7.5 (see Van Wart and Steinbrink, Anal. Biochem. 113, 356 (1981)).

The inactivation studies were carried out as described for enkephalinase and ACE.

Claims

WHAT IS CLAIMED IS:

1. A compound of Formula X

X^-NH-CHR,-(C=0)-X₂

X, wherein X₁₀ is selected from the group consisting of

NC-CHR₂₀-(C=O)- and X₂₁(CR₂₂R_J3) (CHR-,₄) (C=0)-, wherein R₂₀ is hydrogen or alkyl of 1 to 3 carbon atoms, _j,₂, ^ and R^ are each independently hydrogen or alkyl of 1 to 3 carbon atoms, and X₂₁ is fluoro, chloro or bromo; R₉ is benzyl, alkyl of 1 to 5 carbon atoms, or hydrogen; X₂ is joined to the -CO- in an amide linkage and is selected from the group consisting of glycine, N-methyl-glycine, N-benzyl-glycine, D-alanine, L-alanine, 0-alanine, D-phenylalanine, L-phenylalanine, D-leucine, L-leucine, 3-amino propionic acid, D-proline, L-proline, and the group X₃-X₄, wherein X₃ is joined to the -CIH^-CO- group in an amide linkage and is selected from the group consisting of L-proline, L-alanine, L-valine, L-leucine, and L-O-methyltyrosine; when X₃ is L- proline or L-alanine, X₄ is selected from the group consisting of L-arginine, L-proline, L-leucine, L-alanine, L-hydroxyproline, and L-homoarginine; and when X₃ is L- valine, L-leucine, or L-O-methyltyrosine, X₄ is selected from the group consisting of glycine, L-alanine, and the alkyl esters of glycine and alanine, wherein the alkyl is of 1 - 5 carbons; provided that if ^ is ethyl or benzyl, X₂ is not X₃-X₄; if X₂ is X₃-X₄ and X₃ is L-proline or L-alanine, _q is hydrogen or methyl; and if X₂ is X₃-X₄ and X₃ is L- leucine, L-valine or L-O-methyltyrosine, R^ is alkyl of 3 - 5 carbon atoms; and physiologically acceptable salts thereof.

2. A compound according to Claim 1 wherein R₂₀, R₂₂, R₂₃ and R^ are all hydrogen and X₂₁ is chloro.

3. A compound according to Claim 2 wherein, if R_ς is not hydrogen, the configuration of the amino acid residue containing R, is L.

4. A compound according to Claim 3 wherein X₁₀- is Cl(CH₂)₂(CO)-.

5. A compound according to Claim 3 wherein X₁₀- is (N≡C) (CH₂) (C=0)-.

6. A compound according to Claim 4 wherein ^ is benzyl.

7. A compound according to Claim 6 wherein X₂ is selected from the group consisting of glycine, N-methyl-glycine, D-alanine, L-alanine, L-phenylalanine, L-leucine, L-proline and 3-amino propionic acid.

8. A compound according to Claim 5 wherein R₉ is benzyl.

9. A compound according to Claim 8 wherein X₂ is selected from the group consisting of glycine, N-methyl-glycine, D-alanine, L-alanine, L-phenylalanine, L-leucine, L-proline and 3-amino propionic acid.

10. A compound according to Claim 4 wherein R is methyl or hydrogen and X₂ is L-proline.

11. A compound according to Claim 5 wherein R, is methyl or hydrogen and X₂ is L-proline.

12. A compound according to Claim 4 wherein R_ς is hydrogen and X₂ is X₃X₄, wherein X₃ is selected from the group consisting of L-proline and L-valine.

13. A compound according to Claim 4 wherein ^ is isobutyl and X₂ is X₃X₄, wherein X₃ is selected from the group consisting of L-leucine, L-valine and L-O- methyltyrosine.

14. A compound according to Claim 13 wherein X₄ is selected from the group consisting of glycine ethyl ester and L-alanine ethyl ester.

15. A compound according to Claim 5 wherein R₉ is hydrogen and X₂ is X₃X₄, wherein X₃ is selected from the group consisting of L-proline and L-valine.

16. A compound according to Claim 5 wherein R, is isobutyl and X₂ is X₃X₄, wherein X₃ is selected from the group consisting of L-leucine, L-valine and L-O- methyltyrosine.

17. A compound according to Claim 16 wherein X₄ is selected from the group consisting of glycine ethyl ester and L-alanine ethyl ester.

18. A compound of Formula XI X₁₀-NH-CHR₉-(C0₂H)

XI, wherein X₁₀ is selected from the group consisting of N≡C-CHR₂₀-(C=O)- and X₂₁(CR₂₂R₂₃) (CHR₂₄) (C=0)-, wherein R_j-, is hydrogen or alkyl of 1 to 3 carbon atoms, R-,₂, R^ and R₂₄ are each independently hydrogen or alkyl of 1 to 3 carbon atoms, and X₂₁ is fluoro, chloro or bromo; and ₉ is benzyl, alkyl of 1 - 5 carbon atoms, or hydrogen; and salts thereof.

19. A compound according to Claim 1 wherein R₂₀, R₂₂, R_JJ and R^ are all hydrogen and X₂₁ is chloro.

20. A compound according to Claim 3 wherein, if R- is not hydrogen, the configuration of the amino acid residue containing R₉ is L.

21. A compound according to Claim 20 wherein X₁₀- is Cl(CH₂)₂(CO)-.

22. A compound according to Claim 20 wherein X₁₀- is (N≡C) (CH₂) (C=0)-.

23. A compound according to Claim 21 wherein R₉ is benzyl.

24. A compound according to Claim 22 wherein R_g is benzyl.

25. A compound according to Claim 21 wherein , is methyl.

26. A compound according to Claim 22 wherein R is methyl.

27. A compound according to Claim 21 wherein R, is hydrogen.

28. A compound according to Claim 22 wherein ^ is hydrogen.

29. A compound according to Claim 21 wherein ₉ is isobutyl.

30. A compound according to Claim 22 wherein R₉ is isobutyl.

31. A method of reducing pain in a mammal suffering therefrom comprising administering to said mammal a pain-reducing-effective amount of a compound of Formula

wherein X₁₀ is selected from the group consisting of NC-CHR₂₀-(C=O)- and X₂₁(CR^R^) (Cffi^) (C=0)-, wherein R^ is hydrogen or alkyl of 1 to 3 carbon atoms, R₂₂, R^ and R₂₄ are each independently hydrogen or alkyl of 1 to 3 carbon atoms, and X₂₁ is fluoro, chloro or bromo; X₁₂ is joined to the -(C=0)- in an amide linkage and is selected from the group consisting of glycine, N-benzyl-glycine, D-alanine, L-alanine, D-phenylalanine, L-phenylalanine, D-leucine, L-leucine, and 3-amino propionic acid; or a physiologi¬ cally acceptable salt thereof.

32. A method according to Claim 31 wherein the mammal to be treated is a human and, in the compound to be administered, X₁₀- is N≡C-CH₂-(C=0)-, the configuration at the carbon to which the benzyl group is bound is R, and X₁₂ is selected from the group consisting of glycine, D-alanine, L-alanine, L-phenylalanine, L-leucine, and 3-amino propionic acid.

33. A method according to Claim 31 wherein the mammal to be treated is a human and, in the compound to be administered, X₁₀- is C1(CH₂)₂(C=0)-, the configuration at the carbon to which the benzyl group is bound is R, and X₁₂ is selected from the group consisting of glycine,

D-alanine, L-alanine, L-phenylalanine, L-leucine, and 3-amino propionic acid.

34. A method for treating hypertension in a mammal suffering therefrom comprising administering to said mammal an antihypertensive-effective amount of a compound of Formula XV

X₁₀-NH-CHR₁₉-(C=0)-X₁₅

XV, wherein X₁₀ is selected from the group consisting of NC-CHR₂₀-(C=O)- and X₂₁(CR^R^) (CHR^) (C=0)-, wherein R₂₀ is hydrogen or alkyl of 1 to 3 carbon atoms, ^, R^ and R₂₄ are each independently hydrogen or alkyl of 1 to 3 carbon atoms, and X₂₁ is fluoro, chloro or bromo; R₁₉ is benzyl, methyl or hydrogen, X₁₅ is joined to the -(C=0)- in an amide linkage and is selected from the group consisting of glycine, N-methyl-glycine, N-benzyl-glycine, D-alanine, L-alanine, D-phenylalanine, L-phenylalanine, D-leucine, L-leucine, D-proline, L-proline and 3-amino propionic acid; or a physiologically acceptable salt thereof.

35. A method according to Claim 34 wherein the mammal to be treated is a human and, in the compound to be administered, X₁₀- is N≡C-CH₂-(C=0)-, R₁₉ is benzyl, the configuration at the carbon to which the R₁₉ group is bound is R, and X₁₅ is selected from the group consisting of glycine, N-methyl-glycine, L-alanine, L-phenylalanine, L-leucine, and L-proline.

36. A method according to Claim 34 wherein the mammal to be treated is a human and, in the compound to be administered, X₁₀- is C1(CH₂)₂(C=0)-, R₁₉ is benzyl, the configuration at the carbon to which the R₁₉ group is bound is R, and X₁₅ is selected from the group consisting of glycine, N-methyl-glycine, L-alanine, L-phenylalanine, L-leucine, and L-proline.

37. A method according to Claim 34 wherein the mammal to be treated is a human and, in the compound to be administered, X₁₀- is NsC-CH₂-(C=0)-; R₁₉ is methyl or hydrogen; if R₁₉ is methyl, the configuration at the carbon to which the R₁₉ group is bound is R; and X₁₅ is L-proline.

38. A method according to Claim 37 wherein the mammal to be treated is a human and, in the compound to be administered, X₁₀- is C1(CH₂)₂(C=0)-; R₁₉ is methyl or hydrogen; if R₁₉ is methyl, the configuration at the carbon to which the R₁₉ group is bound is R; and X₁₅ is L-proline.

39. A method according to Claim 37 wherein, in the compound to be administered, R₁₉ is hydrogen.

40. A method according to Claim 38 wherein, in the compound to be administered, R₁₉ is hydrogen.

41. A method of making a compound of Formula XX

X₁₀-NH-CHR₂₉-(C=O)-X₂₀

XX, wherein X₁₀ is selected from the group consisting of NC-CHR₂₀-(C=O)- and X₂₁ (CR₂^R_a) (CHR^) (C=0)-, wherein R^ is hydrogen or alkyl of 1 to 3 carbon atoms, ^, ^ and R₂₄ are each independently hydrogen or alkyl of 1 to 3 carbon atoms, and X₂₁ is fluoro, chloro or bromo; RJ_J is benzyl, alkyl of 1 to 5 carbon atoms, or hydrogen; X₂₀ is joined to the -CO- in an amide linkage and is selected from the group consisting of glycine, N-methyl-glycine, N-benzyl-glycine, D-alanine, L-alanine, /3-alanine, D-phenylalanine, L-phenylalanine, D-leucine, L-leucine, 3-amino propionic acid, D-proline, L-proline, and the group X₃₁-X₃₂, wherein X₃₁ is joined to the -CHR^-CO- group in an amide linkage and is selected from the group consisting of L-proline, L-alanine, L-valine, L-leucine, and L-O-methyltyrosine; when X₃₁ is L- proline or L-alanine, X₃₂ is selected from the group consisting of L-arginine, L-proline, L-leucine, L-alanine, L-hydroxyproline, and L-homoarginine; and when X₃₁ is L- valine, L-leucine, or L-O-methyltyrosine, X₃₂ is selected from the group consisting of glycine and L-alanine; provided that if R^ is ethyl or benzyl, X₂₀ is not X₃₁-X₃₂' if X₂₀ is X₃₁-X₃₂ and X₃₁ is L-proline or L-alanine, R₂₉ is hydrogen or methyl; and if X₂₀ is X₃₁-X₃₂ and X₃₁ is L- leucine, L-valine or L-O-methyltyrosine, R^ is alkyl of 3 - 5 carbon atoms; which method comprises: (A) mixed anhydride coupling the compound of Formula XXI

X₁₀-NH-CHR₂₉-(C0₂H)

XXI, wherein X₁₀ and R^ are as defined in the compound of Formula X, with the t-butyl ester of the compound corresponding to X₂₀ in the compound of Formula XX, and (B) acidic hydrolysis of the t-butyl ester made in step (A) .

42. A method of making a compound of Formula XL

X₁₀-NH-CHR₄₉-(C=0)-X₄₀ XL, wherein X₁₀ is selected from the group consisting of NC-CHR₂₀-(C=O)- and X₂₁(CR^R^) (CHR₂₄) (C=0)-, wherein R₂₀ is hydrogen or alkyl of 1 to 3 carbon atoms, R-,₂, R-_g and R₂₄ are each independently hydrogen or alkyl of 1 to 3 carbon atoms, and X₂₁ is fluoro, chloro or bromo; R₄₉ is alkyl of 3 to 5 carbon atoms; and X₄₀ is a dipeptide ester X₄₁X₄₂, wherein X₄₁ is joined to the -CO- in an amide linkage and is selected from the group consisting of L-valine-, L-leucine, or L-O-methyltyrosine, and X₄₂ is selected from the group consisting of the alkyl esters of glycine and L-alanine, wherein the alkyl groups are of 1 - 5 carbon atoms; which method comprises mixed anhydride coupling the compound of Formula XLI

X₁₀-NH-CHR₄₉-(CO₂H) XLI, wherein X₁₀ and R₄₉ are as defined in the compound of Formula XL, with the alkyl ester corresponding to X₄₂ in the compound of Formula XL.

43. The method according to Claim 41 wherein, in the compound of Formula XX, the configuration at the carbon to which R^ is bound, if R_g9 is not hydrogen, is R; and X₁₀ is selected from the group consisting of N≡C(CH₂) (C=0)- and C1(CH₂)₂(C=0)-.

44. The method according to Claim 43 wherein R₂₉ is benzyl.

45. The method according to Claim 42 wherein the configuration at the carbon to which R₄₉ is bound is R; R₄₉ is isobutyl; and X₄₂ is selected from the group consisting of glycine ethyl ester and L-alanine ethyl ester.